General Background on the Value of Nucleic Acid Testing
Applications of nucleic acid testing are broad. The majority of current commercial testing relates to infectious diseases including Chlamydia, gonorrhea, hepatitis and human immunodeficiency virus (HIV) viral load; genetic diseases including cystic fibrosis; coagulation and hematology factors including hemochromatosis; and cancer including genes for breast cancer. Other areas of interest include cardiovascular diseases and drug resistance screening, termed pharmacogenomics. The majority of testing currently occurs in centralized laboratories using non-portable and operationally complex instruments. Presently, tests generally require highly skilled individuals to perform the assays. As a result, the time taken between obtaining a sample suspected of containing a specific nucleic acid fragment and determining its presence or absence is often several hours and even days. However, as with other kinds of blood tests, physicians and others often require results more quickly and obtainable in a convenient user-friendly format. Consequently, there is a need for a portable analysis system capable of performing nucleic acid testing quickly and conveniently. A discussion of prior art relating to various aspects of nucleic acid testing is provided in the following sections.
Methods to Characterize Genetic Information
The clinical manifestation of a particular genetic characteristic can be different with different types or classes of genetic based diseases. This translates into different approaches to measure the genetic characteristic including SNP mutation detection, gene copy mutations and gene overexpression mutations. For example, some diseases such as hemochromatosis, cystic fibrosis or the oncogene p53, have one or a few very specific mutations which affect only a specific nucleotide. Considering hemochromatosis, there are two specific mutations. The clinical manifestation of this disease is an accumulation of iron in various tissues, which can be fatal if untreated. The most prevalent mutation is the G to A transition at nucleotide 845 in the gene, also known as (C282Y). See OMIM: Online Mendelian Inheritance in Man database, which can be found at the U.S. National Center for Biologic Information internet site. The second most prevalent mutation in the same hemochromatosis gene is a C to G transversion in exon 2, known as H63D. These are known as single nucleotide polymorphisms (SNPs). As every individual has two copies of each gene, the possible combinations of these genes are two wild type (homozygous wildtype), two mutated genes (homozygous mutant) or one wild type and one mutated gene (heterozygous). In the case of hemochromatosis, individuals who are homozygous mutant exhibit the disease state, heterozygous individuals can be susceptible for some aspects of the disease as they accumulate higher levels of iron than do homozygous wildtype individuals. Also, for the purpose of determining if an individual is a carrier of the disease to their offspring, the ability to determine that an individual is heterozygous can be useful.
As a result, in testing for a genetic disease like hemochromatosis, it is useful to be able to have at least four analytical means or channels for detection. Here, one channel detects the presence of wildtype C282, a second channel detects the presence of the mutant Y282 gene, a third channel detects the presence of the wildtype H63 gene and the fourth channel detects the presence of the mutant D63 gene. FIG. 12 provides a table of possible outcomes from a hemochromatosis test of this type and shows that it is possible to differentiate between homozygous or heterozygous, and that homozygous channels generate roughly twice the level of expression and thus signal in the test. Note that it is also useful to have one or more additional channels to use as positive and negative controls.
Some genetic mutations include multiple copies of the gene being present in the genome, causing a disease state in a patient. As an example the oncogene ZNF217 mapped within 20q13.2 has been found in multiple copies in individuals with colon cancer (Rooney et al., 2004, J. Pathol. Vol 204 (3):282). Genetic triplication of the alpha-synuclein gene (SNCA) has been reported to cause hereditary early-onset Parkinsonism with dementia (Chartier-Harlin et al., 2004, Lancet, vol 364 (9440):1167). Yamashita et al., 2004, European Neurology, vol 52 (2):101, have found that there is an increase in adult-onset Type III spinal muscular atrophy related to increased gene copies of the survival motor neuron (SMN2) gene. These gene copy mutations can be detected by using one or more required genes, such as the housekeeping genes (e.g. actin or glyceraldehyde-3-phosphate dehydrogenase). Overexpression mutations typically generate increased levels of mRNA and these can be detected.
Methods and Apparatuses for Extraction of Nucleic Acid
Nucleic acids found in cells can be deoxyribonucleic acid or ribonucleic acid and can be genomic DNA, extrachromosomal DNA (e.g. plasmids and episomes), mitochondrial DNA, messenger RNA and transfer RNA. Nucleic acids can also be foreign to the host and contaminate a cell as an infectious agent, e.g. bacteria, viruses, fungi or single celled organisms and infecting multicellular organisms (parasites). Recently, detection and analysis of the presence of nucleic acids has become important for the identification of single nucleotide polymorphisms (SNPs), chromosomal rearrangements and the insertion of foreign genes. These include infectious viruses, e.g. HIV and other retroviruses, jumping genes, e.g. transposons, and the identification of nucleic acids from recombinantly engineered organisms containing foreign genes, e.g. Roundup Ready™ plants.
The analysis of nucleic acids has a wide array of uses. For example, the presence of a foreign agent can be used as a medical diagnostic tool. The identification of the genetic makeup of cancerous tissues can also be used as a medical diagnostic tool, confirming that a tissue is cancerous, and determining the aggressive nature of the cancerous tissue. Chromosomal rearrangements, SNPs and abnormal variations in gene expression can be used as a medical diagnostic for particular disease states. Further, genetic information can be used to ascertain the effectiveness of particular pharmaceutical drugs, known as the field of pharmacogenomics. Genetic variations between humans and between domestic animals can also be ascertained by DNA analysis. This is used in fields including forensics, paternity testing and animal husbandry.
Methods of extracting nucleic acids from cells are well known to those skilled in the art. A cell wall can be weakened by a variety of methods, permitting the nucleic acids to extrude from the cell and permitting its further purification and analysis. The specific method of nucleic acid extraction is dependent on the type of nucleic acid to be isolated, the type of cell, and the specific application used to analyze the nucleic acid. Many methods of isolating DNA are known to those skilled in the art, see for example the general reference Sambrook and Russell, 2001, “Molecular Cloning: A Laboratory Manual”. For example, the prior art contains examples of chemically-impregnated and dehydrated solid-substrates for the extraction and isolation of DNA from bodily fluids that employ lytic salts and detergents and which contain additional reagents for long-term storage of DNA samples e.g. U.S. Pat. No. 5,807,527 detailing FTA paper and U.S. Pat. No. 6,168,922 detailing Isocard Paper. The prior art also contains examples of particle separation methods, e.g. U.S. RE37,891.
Methods of isolating RNA, particularly messenger RNA (mRNA) are well known to those skilled in the art. Typically, cell disruption is performed in the presence of strong protein denaturing solutions, which inactivate RNAses during the RNA isolation procedure. RNA is then isolated using differential ethanol precipitation with centrifugation. As is well known, RNA is extremely labile and is sensitive to alkaline conditions, as well as RNAses, which degrade RNA. RNAses are ubiquitous within the environment and it has been found that they are difficult to remove from solutions and containers used to isolate RNA.
Methods and Apparatuses for Amplification of Nucleic Acid
Polymerase Chain Reaction (PCR) is inhibited by a number of proteins and other contaminants that follow through during the standard methods of purification of genomic DNA from a number of types of tissue samples. It is known that additional steps of organic extraction with phenol, chloroform and ether or column chromatography or gradient CsCl ultracentrifugation can be performed to remove PCR inhibitors in genomic DNA samples from blood. However, these steps add time, complexity and cost. This complexity limits incorporation into a simple disposable cartridge useful for nucleic acid analysis. Therefore, the development of new simple methods to overcome inhibitors found in nucleic acid samples used for nucleic acid amplification processes is desirable.
Nucleic acid hybridization is used to detect discernible characteristics about target nucleic acid molecules. Techniques like the “Southern analysis” are well known to those skilled in the art. Target nucleic acids are electrophoretically separated then bound to a membrane. Labeled probe molecules are then permitted to hybridize to the nucleic acids bound to the membrane using techniques well known in the art. This method is limited, because the sensitivity of detection is dependent on the amount of target material and the specific activity of the probe. As the probe's specific activity may be fixed, to improve the sensitivity of these assays, methods of amplifying nucleic acids are employed. Two basic strategies are employed for nucleic acid amplification techniques; either the number of target copies is amplified, which in turn increases the sensitivity of detection, or the presence of the nucleic acid is used to increase a signal generated for detection. Examples of the first approach are polymerase chain reaction (PCR), rolling circle (see U.S. Pat. No. 5,854,033), and nucleic acid system based amplification (NASBA). Examples of the second include, cycling probe reaction, termed CPR (see U.S. Pat. No. 4,876,187 and U.S. Pat. No. 5,660,988) and SNPase assays, e.g. the Mismatch Identification DNA Analysis System (see U.S. Pat. No. 5,656,430 and U.S. Pat. No. 5,763,178).
The PCR reaction is well known to those skilled in the art and was originally described in U.S. Pat. No. 4,683,195. The process involves denaturing nucleic acid, a hybridization step and an extension step in repeated cycles and is performed by varying the temperature of the nucleic acid sample and reagents. This process of subjecting the samples to different temperatures can be effected by placing tubes into different temperature water baths, or by using peltier-based devices capable of generating heating or cooling, dependent on the direction of the electrical current as described in U.S. Pat. No. 5,333,675 and U.S. Pat. No. 5,656,493. Many commercial temperature cycling devices are available, sold for example by Perkin Elmer, Applied Biosystems and Eppendorf. As these devices are generally large and heavy they are not generally amenable to use in non-laboratory environments, e.g. at the point-of-care.
A microfabricated device for performing the polymerase chain reaction is described in U.S. Pat. No. 5,639,423 though it is silent on providing an integrated means for extracting nucleic acids. A device for performing the polymerase chain reaction is described in U.S. Pat. No. 5,645,801 which has an amplification chamber that can be mated in a sealable manner to a chamber for detection. U.S. Pat. No. 5,939,312 describes a miniaturized multi-chamber polymerase chain reaction device. U.S. Pat. No. 6,054,277 describes a silicon-based miniaturized genetic testing platform for amplification and detection. A polymer-based heating component for amplification reactions is described in U.S. Pat. No. 6,436,355. U.S. Pat. No. 6,303,288 describes an amplification and detection system with a rupturable pouch containing reagents for amplification. U.S. Pat. No. 6,372,484 describes an apparatus for performing the polymerase chain reaction and subsequent capillary electrophoretic separation and detection in an integrated device.
There are several nucleic acid amplification technologies that differ from the PCR reaction in that the reaction is run at a single temperature. These isothermal methods include the cycling probe reaction, strand displacement, Invader™, SNPase, rolling circle reaction and NASBA. U.S. Pat. No. 6,379,929 describes a device for performing an isothermal nucleic acid amplification reaction.
More recently, a strategy for performing the polymerase chain reaction isothermally has been described by Vincent et al., 2004, EMBO Reports, vol 5 (8), see also US Application 20040058378. A DNA helicase enzyme is used to overcome the limitations of heating a sample to perform PCR DNA amplification.
Enzymes Used for the Polymerase Chain Reaction (PCR)
The polymerase chain reaction (PCR) is based on the ability of a DNA polymerase enzyme to exhibit several core features, which include its ability to use a primer sequence with a 3′-hydroxyl group and a DNA template sequence and to extend a newly synthesized strand of DNA using the template strand, all well known to those skilled in the art. In addition, DNA polymerases used in the PCR reaction must be able to withstand high temperatures (e.g. 90 to 99° C.) used to denature double stranded DNA templates, as well as be inactive at lower temperatures (e.g. 40 to 60° C.) at which DNA primers hybridize to the DNA template. Further, to have optimal DNA synthesis at a temperature near to the hybridization temperature (e.g. 60 to 80° C.).
In addition to these core characteristics, DNA polymerases also exhibit proofreading capabilities, which are due to the 3′-5′ exonuclease activity inherent in most DNA polymerases. For the purpose of single nucleotide polymorphism (SNP) detection based on differential primer extension using PCR (also called 3′-allele specific primer extension), it is a disadvantage to use an enzyme that exhibits a 3′-5′ exonuclease activity, as the terminal 3′ nucleotide can be excised from a standard nucleic acid primer, permitting synthesis of both alleles.
Zhang et al., (2003, Laboratory Investigation, vol 83(8):1147) describe the use of a terminal phosphorothioate bond to overcome the limitations of DNA polymerases used for 3′-5′ exonuclease activity. The phosphorothioate bond is not cleaved by 3′-5′ exonucleases. This prevents DNA polymerases with 3′-5′ exonuclease activities from removing the terminal mismatch and proceeding with DNA elongation, alleviating the lack of discrimination observed with normal DNA.
Another characteristic of DNA polymerases is their elongation rate. Takagi et al., (1997, Applied and Environmental Microbiology, vol 63 (11): 4504) teach that Pyrococcus sp. Strain KOD1 (now Thermococcus kodakaraensis KOD1), Pyrococcus furiosus, Deep Vent (New England Biolabs, Beverly, Mass.), and Thermus aquaticus have elongation rates of 106 to 138, 25, 23 and 61 bases/second, respectively. The processivity rates of these enzymes are also described, and behave similarly to the elongation rates. Clearly, Thermococcus kodakaerensis KOD1 has much higher elongation and processivity rates compared to the other well-known enzymes, which would make this enzyme beneficial in applications where sensitivity and speed are an issue. Further, Thermococcus kodakaerensis KOD1 possesses an exonuclease activity which would be detrimental for use in a 3′-allele specific primer extention assay used for SNP analysis.
Design of Synthetic Oligonucleotides
Regarding the design of synthetic oligonucleotides for use in amplification reactions, Rychlik et al., (1989, Nucleic Acids Research, vol 17 (21):8543-8551) and Rychlik (1995, Molecular Biotechnology, vol 3: 129-134), describe selection criteria and computer programs to design probes and primers, including primers for in vitro amplification of DNA. Both teach that primers should not generate secondary structure or exhibit self-hybridization.
PCR primers designed as molecular beacons (Bonnet et al., 1999, Proc. Natl. Acad. Sci. USA, vol 96: 6171-6176) have a short region at both the 5′ and 3′ ends which are complementary generating what is known as hairpin loop structures, to quench the fluorescent signal by placing the donor and quencher molecules in close physical proximity to each other. After polymerization and incorporation into a newly synthesized double stranded molecule, the donor and quencher molecules are physically distant to each other, permitting the generation of a fluorescent signal. The region of complementarity is short and typically has only about 5 nucleotides which are complementary, preferably generating a hairpin stem. Tsourkas et al., 2003, Nucleic Acids Research, vol 31 (4):1319-1330, teaches that molecular beacons with longer stem lengths have an improved ability to discriminate between targets over a broader range of temperatures. However, this is accompanied by a decrease in the rate of molecular beacon-target hybridization. Molecular beacons with longer probe lengths tend to have lower dissociation constants, increased kinetic rate constants and decreased specificity. Therefore, having longer stem loops will have an impact on reducing the efficiency of hybridization kinetics, which in turn will reduce the levels of PCR amplification. Therefore, PCR using a stem loop structure is generally undesirable in the art. Kaboev et al., (2000, Nucleic Acids Research, vol 28 (21):e94) teaches that designing a PCR primer with a stem loop structure by adding additional sequences to the 5′-end of the primer, which are complementary to the 3′-end. This reference also teaches that adding this secondary structure increases the specificity of the PCR reaction, though it does use a PCR primer that permits the generation of single stranded tails. Further, Kaboev does not teach that the generation of the secondary structure prevents the hybridization of the single stranded regions to a capture moiety.
Detection Methods
Conventional detection methods for the final step in a nucleic acid analysis are well known in the art and include sandwich-type capture methods based on radioactivity, colorimetry, fluorescence, fluorescence resonance energy transfer (FRET) and electrochemistry. For example, jointly owned U.S. Pat. No. 5,063,081 covers a sensor for nucleic acid detection. The sensor has a permselective layer over an electrode and a proteinaceous patterned layer with an immobilized capture oligonucleotide. The oligonucleotide can be a polynucleotide, DNA, RNA, active fragments or subunits or single strands thereof. Coupling means for immobilizing nucleic acids are described along with methods where an immobilized nucleic acid probe binds to a complimentary target sequence in a sample. Detection is preferably electrochemical and is based on a labeled probe that also binds to a different region of the target. Alternatively, an immobilized antibody to the hybrid formed by a probe and polynucleotide sequence can be used along with DNA binding proteins. The '081 patent incorporates by reference the jointly owned patent U.S. Pat. No. 5,096,669 which covers a single-use cartridge for performing assays in a sample using sensors. These sensors can be of the type described in '081.
Other divisional patents related to '081 include U.S. Pat. No. 5,200,051 which covers a method of making a plurality of sensors with a permselective membrane coated with a ligand receptor that can be a nucleic component. U.S. Pat. No. 5,554,339 covers microdispensing, where a nucleic acid component is incorporated into a film-forming latex or a proteinaceous photoformable matrix for dispensing. U.S. Pat. No. 5,466,575 covers methods for making sensors with the nucleic component incorporated into a film-forming latex or a proteinaceous photoformable matrix. U.S. Pat. No. 5,837,466 covers methods for assaying a ligand using the sensor components including nucleic components. For example, a quantitative oligonucleotide assay is described where the target binds to a receptor on the sensor and is also bound by a labeled probe. The label is capable of generating a signal that is detected by the sensor, e.g. an electrochemical sensor. U.S. Pat. No. 5,837,454 covers a method of making a plurality of sensors with a permselective membrane coated with a ligand receptor that can be a nucleic component. Finally, jointly owned U.S. Pat. No. 5,447,440 covers a coagulation affinity-based assay applicable to nucleotides, oligonucleotides or polynucleotides. These jointly owned patents are incorporated herein by reference.
It is noteworthy that jointly owned U.S. Pat. No. 5,609,824 discloses a thermostated chip for use within a disposable cartridge applicable to thermostating a sample, e.g. blood, to 37° C. Jointly owned U.S. Pat. No. 6,750,053 and pending US 20030170881 address functional fluidic elements of a disposable cartridge relevant to various tests including DNA analyses. These additional jointly owned patents and applications are incorporated herein by reference.
Several other patents address electrochemical detection of nucleic acids, for example U.S. Pat. No. 4,840,893 discloses detection with an enzyme label that uses a mediator, e.g. ferrocene. U.S. Pat. No. 6,391,558 discloses single stranded DNA on the electrode that binds to a target, where a reporter group is detected by the electrode towards the end of a voltage pulse and uses gold particles on the electrode and biotin immobilization. U.S. Pat. No. 6,346,387 discloses another mediator approach, but with a membrane layer over the electrode through which a transition metal mediator can pass. U.S. Pat. No. 5,945,286 is based on electrochemistry with intercalating molecules. U.S. Pat. No. 6,197,508 discloses annealing single strands of nucleic acid to form double strands using a negative voltage followed by a positive voltage. Similar patents include U.S. Pat. No. 5,814,450, U.S. Pat. No. 5,824,477, U.S. Pat. No. 5,607,832 and U.S. Pat. No. 5,527,670 which disclose electrochemical denaturation of double stranded DNA. U.S. Pat. No. 5,952,172 and U.S. Pat. No. 6,277,576 disclose DNA directly labeled with a redox group.
Several patents address devising cartridge-based features or devices for performing nucleic acid analyses, these include for example a denaturing device U.S. Pat. No. 6,485,915, an integrated fluid manipulation cartridge U.S. Pat. No. 6,440,725, a microfluidic system U.S. Pat. No. 5,976,336 and a microchip for separation and amplification U.S. Pat. No. 6,589,742.
Based on the forgoing description there is a need for a convenient and portable analysis system capable of performing nucleic acid testing.